This procedure aims to identify metabolites transferred from sperm to oocytes during fertilization by using stable isotope labeling with 2H2O or U13C Glucose followed by metabolomic analysis. Deuterium-labeled sires are mated with unlabeled dams. Collected pre-implantation embryos are analyzed by mass spectrometry to sort paternal (deuterated) from maternal (non-deuterated) metabolites.
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Method Article
Stable Isotope In-Vivo Labeling for Mass-Spectrometry Identification of Paternal Metabolites Transferred from Sperm to Oocyte During Fertilization
In This Article
Summary
Abstract
Paternal contribution to embryo genetics has so far been limited to allelic sequences for decades. A decade of results suggests, instead, that epigenetic factors-DNA methylation, histone modifications, chromosomal organization, and regulatory RNAs-play crucial roles in paternal inheritance and influence embryonic development and zygote gene expression. Together with nucleic acids, sperm metabolome (lipids, carbohydrates, free amino acids) may act as epigenetic signal. This protocol aims to identify sperm-to-oocyte transferred metabolites by mass spectrometry. The procedure includes stable isotope labeling of sperm cells with 2H2O or U13C Glucose to trace, by metabolomics, paternal metabolites transferred to the oocyte during fertilization. The overall goal of the protocol is to reveal the role of paternal sperm metabolome in offspring's susceptibility to dysmetabolism, and it may be adapted to provide insights into how environmental conditions, such as diet and exposure to pollutants, alter paternal metabolic messages, potentially affecting offspring's health and predisposition to diabetes, obesity, and cardiovascular diseases.
Introduction
Paternal epigenome influences offspring's susceptibility to dysmetabolism
Differently from the plethora of pre- and post-conceptional roles exploited by mothers during embryo development, the contribution of sperm cells has been thought for decades to be exclusively genetic. According to genetic inheritance theory, paternal contribution to the phenotypic variability of an individual (including its susceptibility to disease) had to be ascribed exclusively to the genetic sequence of paternally inherited allelic variants and to their interactions with those maternally inherited1. Nowadays, it is clear that alleles are no....
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Protocol
All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Naples Federico II, Italy. The reagents and the equipment used in this study are listed in the Table of Materials.
1. Selection and grouping of C57BL/6J males
- Select C57BL/6J males of about 12 weeks.
- Divide males into two groups and name them LABELED ( undergoing stable isotope labeling) or UNLABELED (used as control and for measuring the efficacy of isotope labeling in sperm cells and morulae).
- In order to empty the epididymis from unlabeled sperm cells, allow the mal....
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Results
In order to monitor metabolite transfer from sperm to oocyte, morulae isolated from C57 BL/6J females mated with sires from LABELED and UNLABELED groups are collected and analyzed by Mass-Spectrometry based Metabolomics. Metabolites were extracted as described in steps 6-7 and derivatized with TMS as described in step 8. Metabolomic and Lipidomic profiling of LABELED morulae, UNLABELED morulae, UNLABELED oocytes, LABELED, and UNLABELED sperm cells was performed. The resulting profiles were statistically analyzed, aligned.......
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Discussion
The described protocol allows the identification of metabolites transferred from sperm to oocytes during fertilization by using stable isotope labeling of male mice with 2H2O or U13C Glucose followed by metabolomic analysis. Isotopically labeled metabolites found in morulae are of paternal origin.
Although hydrogen and deuterium are isotopes, they are not entirely identical in terms of chemical behavior. The melting and boiling points of 2H2
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Disclosures
The authors declare no competing interests.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 2H2O filtered, 99.8 atom % 2H | Sigma Aldrich | 756822 | |
| Acetone | Sigma Aldrich | 179124 | |
| Agilent SIL-8 | Agilent (Santa Clara, CA, U.S.A.) | ||
| C16-Ceramide-13C3-15N | LGC standard | TRC-C342770-25MG | |
| dichloromethane | Sigma Aldrich | 34856 | |
| Diethyl ether | Sigma Aldrich | 673811 | |
| ethanol | Sigma Aldrich | 1,11,727 | |
| HCG (Human chorionic gonadotropin, | Sigma Aldrich | CG5-1VL | |
| L-Lysine-ε-15N hydrochloride | Sigma Aldrich | 608971 | |
| Male and female C57BL/6JOlaHsd mice | Inotiv | C57BL/6JOlaHsd | |
| methyl tert-butyl ether (MTBE) | Sigma Aldrich | 306975 | |
| MetOH | Sigma Aldrich | 1,06,035 | |
| N,O-Bis(trimethylsilyl(TMS) trifluoroacetamide (BSTFA) | Supelco | 15238 | |
| PBS | Applichem | A0965-9010 | |
| PMSG (Pregnant mare’s serum) | Lee Biosolutions | 493-10 | |
| pyridine | Sigma Aldrich | 360570 | |
| Shimadzu GCMS 2010plus | Shimadzu | ||
| Sitostanol-5,6,22,23-d4 | Sigma Aldrich | 680028 | |
| sodium trifluoracetate | Sigma Aldrich | 1615138 | |
| SolariX XR 7T | Bruker Daltonics | ||
| TimsTOF Pro Q-TOF | Bruker Daltonics | ||
| U13C glucose | Sigma Aldrich | 389374 | |
| Ultimate RS3000 UHPLC | Thermo Fisher Scientific | ||
| VanGuard CSHTM precolumn (5.0 × 2.1 mm; 1.7 μm, 130 Å) | Waters |
References
- Wang, Y., Liu, H., Sun, Z. Lamarck rises from his grave: Parental environment-induced epigenetic inheritance in model organisms and humans. Biol Rev. 92 (4), 2084-2111 (2017).
- Champroux, A., Cocquet, J., Henry-Berger, J., Drevet, J. R., Kocer, A.
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